CN103891713A - Anti-browning sanitizer for plant tissue culture explant - Google Patents
Anti-browning sanitizer for plant tissue culture explant Download PDFInfo
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- CN103891713A CN103891713A CN201410113999.0A CN201410113999A CN103891713A CN 103891713 A CN103891713 A CN 103891713A CN 201410113999 A CN201410113999 A CN 201410113999A CN 103891713 A CN103891713 A CN 103891713A
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- China
- Prior art keywords
- explant
- disinfectant
- tissue culture
- benzyl
- chlorophenol
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- 238000004161 plant tissue culture Methods 0.000 title claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- NCKMMSIFQUPKCK-UHFFFAOYSA-N 2-benzyl-4-chlorophenol Chemical compound OC1=CC=C(Cl)C=C1CC1=CC=CC=C1 NCKMMSIFQUPKCK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000002791 soaking Methods 0.000 claims abstract description 5
- 238000005406 washing Methods 0.000 claims abstract description 5
- 239000000645 desinfectant Substances 0.000 claims description 26
- 238000004659 sterilization and disinfection Methods 0.000 claims description 26
- 239000008223 sterile water Substances 0.000 claims description 15
- RDBMUARQWLPMNW-UHFFFAOYSA-N phosphanylmethanol Chemical compound OCP RDBMUARQWLPMNW-UHFFFAOYSA-N 0.000 claims description 12
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 claims description 11
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 230000000844 anti-bacterial effect Effects 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 239000003899 bactericide agent Substances 0.000 claims description 2
- GQCLFTGMXCYYHT-UHFFFAOYSA-N phosphanium;hydroxymethyl sulfate Chemical compound [PH4+].OCOS([O-])(=O)=O GQCLFTGMXCYYHT-UHFFFAOYSA-N 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 25
- 230000001954 sterilising effect Effects 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 12
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 abstract description 11
- 239000005708 Sodium hypochlorite Substances 0.000 abstract 1
- ZGTNBBQKHJMUBI-UHFFFAOYSA-N bis[tetrakis(hydroxymethyl)-lambda5-phosphanyl] sulfate Chemical compound OCP(CO)(CO)(CO)OS(=O)(=O)OP(CO)(CO)(CO)CO ZGTNBBQKHJMUBI-UHFFFAOYSA-N 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 abstract 1
- 229960002523 mercuric chloride Drugs 0.000 abstract 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 abstract 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 241000196324 Embryophyta Species 0.000 description 9
- 230000007613 environmental effect Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 239000003963 antioxidant agent Substances 0.000 description 5
- 230000003078 antioxidant effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000011575 calcium Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 241001505935 Phalaenopsis Species 0.000 description 3
- 244000127818 Phalaenopsis amabilis Species 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 150000002989 phenols Chemical class 0.000 description 3
- 231100000614 poison Toxicity 0.000 description 3
- 230000007096 poisonous effect Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000195940 Bryophyta Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- -1 clorox (saturated calcium hypochlorite Chemical class 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000002070 germicidal effect Effects 0.000 description 2
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
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- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 1
- IXVMHGVQKLDRKH-YEJCTVDLSA-N (22s,23s)-epibrassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@H](O)[C@@H](O)[C@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-YEJCTVDLSA-N 0.000 description 1
- ISPYQTSUDJAMAB-UHFFFAOYSA-N 2-chlorophenol Chemical compound OC1=CC=CC=C1Cl ISPYQTSUDJAMAB-UHFFFAOYSA-N 0.000 description 1
- SXXLKZCNJHJYFL-UHFFFAOYSA-N 4,5,6,7-tetrahydro-[1,2]oxazolo[4,5-c]pyridin-5-ium-3-olate Chemical compound C1CNCC2=C1ONC2=O SXXLKZCNJHJYFL-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 240000000595 Aglaia odorata Species 0.000 description 1
- 235000010887 Aglaia odorata Nutrition 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 description 1
- 101100433727 Caenorhabditis elegans got-1.2 gene Proteins 0.000 description 1
- 235000005881 Calendula officinalis Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000004875 Carya cathayensis Species 0.000 description 1
- 235000005663 Carya cathayensis Nutrition 0.000 description 1
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 1
- 229910052684 Cerium Inorganic materials 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 241001517183 Epidendrum Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 101000799461 Homo sapiens Thrombopoietin Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 240000003553 Leptospermum scoparium Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 229910052779 Neodymium Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 240000005001 Paeonia suffruticosa Species 0.000 description 1
- 235000003889 Paeonia suffruticosa Nutrition 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 229910052777 Praseodymium Inorganic materials 0.000 description 1
- 244000178231 Rosmarinus officinalis Species 0.000 description 1
- 229910052772 Samarium Inorganic materials 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 240000000785 Tagetes erecta Species 0.000 description 1
- 102100034195 Thrombopoietin Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 239000012984 antibiotic solution Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 1
- 235000005487 catechin Nutrition 0.000 description 1
- ZMIGMASIKSOYAM-UHFFFAOYSA-N cerium Chemical compound [Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce] ZMIGMASIKSOYAM-UHFFFAOYSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229950001002 cianidanol Drugs 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 235000020197 coconut milk Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 description 1
- BEUKRGSOWSZCQH-UHFFFAOYSA-N hydroxymethyl hydrogen sulfate Chemical compound OCOS(O)(=O)=O BEUKRGSOWSZCQH-UHFFFAOYSA-N 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 239000008235 industrial water Substances 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229910052746 lanthanum Inorganic materials 0.000 description 1
- FZLIPJUXYLNCLC-UHFFFAOYSA-N lanthanum atom Chemical compound [La] FZLIPJUXYLNCLC-UHFFFAOYSA-N 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- QEFYFXOXNSNQGX-UHFFFAOYSA-N neodymium atom Chemical compound [Nd] QEFYFXOXNSNQGX-UHFFFAOYSA-N 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- HOKBIQDJCNTWST-UHFFFAOYSA-N phosphanylidenezinc;zinc Chemical compound [Zn].[Zn]=P.[Zn]=P HOKBIQDJCNTWST-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- PUDIUYLPXJFUGB-UHFFFAOYSA-N praseodymium atom Chemical compound [Pr] PUDIUYLPXJFUGB-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
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- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- NGOCMUBXJDDBLB-UHFFFAOYSA-N trifluoromethanesulfonic acid;zinc Chemical compound [Zn].OS(=O)(=O)C(F)(F)F NGOCMUBXJDDBLB-UHFFFAOYSA-N 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an anti-browning sanitizer for a plant tissue culture explant, and belongs to plant tissue culture technologies in agriculture and forestry fields. A method in which the surface of the explant is sterilized with a reducible sterilant which replaces conventional mercuric chloride or sodium hypochlorite comprises steps of washing the tissue culture explant with flowing water, then sterilizing instantly with an ethanol solution, and soaking the explant respectively through tetrakis hydroxymethyl phosphonium sulfate and a 2-benzyl-4-chlorophenol solution in sequence. Compared with a conventional explant sanitizer, the reducible sterilant is efficient, has the broad spectrum, is mild to plant cells, inhibits browning while sterilizing the explant, improves the pretreatment efficiency of the explant, lowers the death rate of the tissue culture explant, is simple and convenient to operate, is safe to people, is convenient in popularization and application and has the environment-friendly effect.
Description
Technical field
The invention belongs to the plant tissue culture technique in agricultural field, be specifically related to the sterilization of group training explant and anti-brownization method.
Technical background
The production of plant tissue culture technique seedling, Virusfree, germplasm improvement etc. have wide market prospects.Also there are many problems in plant tissue culture technique link at present, and the control that explant pollutes is that tissue is cultivated successful Major Difficulties.Conventional explant sterilization method is first to use 70% alcohol-pickled 10-30s, and with aseptic water washing 2-3 time, 0.1% mercury chloride soaks 8-12min or with 2% clorox (saturated calcium hypochlorite solution) immersion 15-20min.Although mercury chloride Disinfection Effect is good, trace volatilization under normal temperature, skin-permeable, extremely strong to people's toxicity, lethal dose is 0.05 gram, large to environmental hazard, in October, 2013 China's signature " Minamata mercury pollution is prevented and treated pact ", the production application of mercurial will more sternly be controlled.Clorox (calcium) thus emit Active Chlorine ion and kill thalline, Disinfection Effect is poor compared with mercury chloride, the chlorine of generation stimulates large to human body, poisonous, liquor natrii hypochloritis's oxidisability is very strong, and vegetable material is had to larger destruction lethal effect.In addition, use antibiotic sterilization, bactericidal range is narrow, and antibiotic solution needs filtration sterilization, and plant easily develops immunity to drugs, and also easily causes material physiological variation.
Still first-selected mercury chloride and clorox of the explant sterilization of group training at present.The explant disinfectant patent of not mercurous or clorox (calcium) " bryophyte is as the purposes of plant explants disinfectant and this antiseptic composition and preparation method thereof and using method " (publication No.: 101632392), disinfectant composition is bryophyte extract, also comprise bleeding agent, stabilizing agent and cosolvent, but disinfectant raw material are not universal, extraction procedure is loaded down with trivial details, cost is high, may will limit the wide popularization and application of this invention.Patent " surface sterilizing method of explant " (publication No.: 101461329), with sterilization explant before and after alcohol, bromine water, bromine water has stronger oxidative damage to plant, and Disinfection Effect is general, is not first-selected disinfectant.And other still use mercury chloride or clorox about explant disinfectant patent, as publication No. 1628507A, 101138317A, 102326514A, 101946809A.
Brown is also that tissue is cultivated one of successful Three Obstacles, and the method that overcomes Brown mainly contains: (1) selects suitable explant collection season and position.(2) antioxidant soaks explant.(3) medium adds antioxidant, activities of antioxidant enzymes promoter and phenols (pigment) material adsorbent.(4) culture is repeatedly transferred to fresh culture.The antioxidant that wherein (2) and (3) generally adopt mainly contains ascorbic acid, cysteine, citric acid, sulphite.The material brassinosteroid that strengthens activities of antioxidant enzymes in plant explants, phenols (pigment) material adsorbent has polyvinylpyrrolidone or active carbon.As patent publication No. CN103250645A, CN102986534, CN102986533, CN103004754A, CN102805032A, CN102823490A, CN102577953A, CN103125380A, CN102318560A, CN102860255A, CN102115727A.
More distinctive Browning control patent has: publication No. CN103202233A " a kind of Moth orchid anti-browning tissue culture method and anti-browning medium " adds marigold, lemon, tomato, rosemary, Xue MingRosma rinus officinalis etc. extract in corresponding medium in first culture and Proliferation, Differentiation cultivation.CN103004602A " a kind of method that suppresses cathay hickory Brown " catechin is applied in medium.CN102870677A " a kind of promote narrow leaved tea tree adventitious buds proliferation and prevent that subculture from cultivating the method for brownization " medium application rare earth compound comprises nitrate and the oxide of lanthanum, cerium, neodymium, praseodymium, samarium.CN101148653 " suppress epidendrum tissue and cultivate the method that brown stain occurs " by after explant sterilization with gibberellin and/auxin soaking, evoking adventive bud adds 6-BA at medium, 4-PU or TDZ.CN1737142 " a kind of method that prevents that plant callus browning from producing " add polyamines (spermine or spermidine) at subculture medium, dark culturing, the regular gas exchange of blake bottle.The MS medium that CN1742564 " a kind of tree peony group training seedling proliferation and brownization and vitrifying prevention method " adopts is got 1/2-1/3 trace element, Ca
2+double CaCl
2with CaNO
3mol ratio be 1: 1.
At present sterilize first-selected mercury chloride or clorox (calcium) of the external body of plant tissue culture, has larger toxic and side effect to explant, poisonous to human body, harm environment, and explant disinfection and anti-brownization substep carry out, inefficiency.Be badly in need of research and development sterilization broad-spectrum high efficacy, little to phyto toxicity, to human-body safety, environmental friendliness, easy to operate, low-cost new disinfectant.
Summary of the invention
What the present invention solved is that current Plant Tissue Breeding explant disinfectant is poisonous to human body, serious environment pollution and plant culture is injured to large technical problem.Due to reducing substances to plant cell action temperature and; damage little; can also suppress explant otch aldehydes matter and be oxidized to noxious material (quinones); thereby protective plant cell; so Browning control when adopting reproducibility efficient germicide to have the sterilization of group training explant, the double effects of raising survival rate.The present invention has announced composition, compound method and the using method of anti-brownization disinfectant.
The anti-brownization disinfectant of a kind of plant tissue culture explant and sterilization method, is characterized in that the anti-brownization disinfectant tetrakis hydroxymetyl phosphonium sulfuric (THPS) adopting, molecular formula [P (CH2OH)
4]
2sO
4can comparatively fast kill cause of disease (carefully) bacterium, for environment-friendly type disinfection of indoor air, water treatment biocide, and it has stronger reproducibility, in environment, can be oxidized to soon the THPO that toxicity is extremely low, can directly discharge (Zhang Jing, the yellow race buys. the friendly biocide-tetrakis hydroxymetyl phosphonium sulfuric of a kind of novel environmental. and Treatment of Industrial Water, 2004,24 (11): 13-15; Intelligence is built brightness, Tang Bin, Li Lifang etc. disinfection of indoor air effect and the toxicity of tetra methylol sulfuric acid phosphine. and environment and health magazine, 2007,24 (3): 156-158; Yang Wenzhong, green Water Treatment Chemicals. meticulous and specialty chemicals 2007.15 (23): 17-19; Yang Wei compiling. the friendly bactericide tetra methylol sulfuric acid squama of novel environmental (THPS). phosphate industry, 2003 (2) 10-7).
Another anti-brownization disinfectant 2-benzyl-4-chlorophenol that the present invention announces, C13H11ClO, phenols disinfectant, reproducibility is strong, and fungi, bacterium are had to better kill activity, can be used as agricultural chemicals, medicine intermediate.That Environmental Protection Agency notices the component that allows to can be used for food contacting surface cleaning agent, also be the component that U.S. FDA approval can be used for food processing machinery and container sterilization, environmental friendliness, in water, degradability reaches more than 94% (Han Haijun, the synthesising process research of the chloro-phenol of the pretty .2-benzyl-4-of burnt family. Gansu petroleum and chemical industry, 2006 (2): 15-18; Ma Zhonghua, Zhang Jiayan, Nie Jin. trifluoromethane sulfonic acid zinc catalytic selectivity synthesizes 2-benzyl-4-chlorophenol. applied chemistry, 2010,27 (3): 304-307).
Technical scheme provided by the invention is as follows:
Described anti-brownization disinfectant tetrakis hydroxymetyl phosphonium sulfuric is directly dissolved in water, is made into the concentration 0.5-1.0% aqueous solution.2-benzyl-4-chlorophenol is dissolved in absolute ethyl alcohol and Tween-20, than being 1: 3: 5, is then dissolved in water with ethanol, Tween-20 weight (g) volume (ml), 2-benzyl-4-chlorophenol working concentration 0.5%.
Technical scheme provided by the invention also comprises that disinfectant applied in any combination is in the using method of explant sterilization.Conventional method obtains explant, after flowing water rinses, 75% ethanol soaks 10-60 second, soaks 60-90 minute through tetrakis hydroxymetyl phosphonium sulfuric of the present invention, repeatedly stir during this time or concussion, rinsed with sterile water 3 times, 2-benzyl-4-chlorophenol solution soaks 30-60 minute, repeatedly stirs during this time or concussion, without rinsed with sterile water, make the medicament that remains in explant can continue to play a role, only blot explant surface solution with filter paper, just can conventional inoculate.
Beneficial effect of the present invention is: soak explant with reproducibility efficient germicide, fungi, bacterium are had to very fast killing action, can reduce explant pollution rate, also can reduce brown rate, improve survival rate, odorlessness under this medicament working concentration, harmless, environmental friendliness, with low cost, suitablely promote in field of plant tissue culture technique.
Embodiment
Embodiment mono-
More than Moth orchid (Phalaenopsis spp.) leaf stripping and slicing flowing water 30 rinses minute, in 75% 30 seconds of ethanol, sterile water washing 2 times, uses respectively following methods soaking disinfection (1) 2% clorox to soak 30 minutes, rinsed with sterile water 3 times.(2) 0.1% mercury chloride soak 8 minutes, rinsed with sterile water 5 times.(3) 0.5% tetrakis hydroxymetyl phosphonium sulfuric solution soak 60 minutes, and after rinsed with sterile water 3 times, 2-benzyl-4-chlorophenol solution of 0.5% soaks 30 minutes, and without rinsed with sterile water, filter paper blots surface solution, directly inoculation.Medium component 1/2MS+6-BA5mg/L+NAA0.2mg/L+ coconut milk 20% (V/V).6 explants of every bottle graft kind,, repeat 3 times by each 5 bottles.After inoculation, under 25 DEG C, 1500lux light, cultivate illumination 12h/d.
Phalaenopsis Explant in Vitro was cultivated after 29 days, minimum with disinfectant of the present invention and 0.1% mercury chloride sterilization pollution rate, was all 0, and what clorox was sterilized has a small amount of pollution.The brown rate of disinfectant processing of the present invention is lower than clorox sterilization, than much lower with mercury chloride, in table 1, so disinfectant of the present invention has application advantage in Phalaenopsis leaves sterilization, is suitable disinfectant.
The each sterilization method effect observation of table 1 Moth orchid leaf stripping and slicing explant *
* the explant of brownization refers to that brownization area exceedes explant more than explant half of the area.Brown rate is with uncontaminated explant observation statistics.
Embodiment bis-
There is stem segment in Milan (Aglaia odorata), and flowing water rinses 2 hours, and in 75% 30 seconds of ethanol, sterile water washing 2 times, uses respectively following methods soaking disinfection (1) 2% clorox to soak 30 minutes, rinsed with sterile water 3 times.(2) 0.1% mercury chloride soak 12 minutes, rinsed with sterile water 5 times.(3) 1% tetrakis hydroxymetyl phosphonium sulfuric solution soak 60 minutes, rinsed with sterile water 3 times, and 2-benzyl-4-chlorophenol solution of 0.5% soaks 60 minutes, and without rinsed with sterile water, filter paper blots surface solution, directly inoculation.(4) 0.5% tetrakis hydroxymetyl phosphonium sulfuric solution soak 60 minutes, rinsed with sterile water 3 times, and 2-benzyl-4-chlorophenol solution of 0.5% soaks 40 minutes, and without rinsed with sterile water, filter paper blots surface solution, directly inoculation.
Explant inoculated and cultured based component MS+6-BA2mg/L+NAA0.2mg/L.6 explants of every bottle graft kind,, repeat 3 times by each 5 bottles.After inoculation, under 25 DEG C, 1500lux light, cultivate, illumination 12h/d, the results are shown in Table 2.
There is the each sterilization method effect observation of stem segment explant * in table 2 Milan
* the explant of brownization refers to that brownization area exceedes explant more than explant half of the area.Brown rate is with uncontaminated explant observation statistics.
Milan explant was cultivated after 30 days, and it is slightly lower than mercury chloride that 1.0% tetrakis hydroxymetyl phosphonium sulfuric+0.5%2-benzyl-4-chlorophenol is processed pollution rate, and brown rate significantly reduces, and is better than mercury chloride result of use, recommendation.It is too high that clorox, 0.5% tetrakis hydroxymetyl phosphonium sulfuric+0.5%2-benzyl-4-chlorophenol are processed pollution rate, not to recommend.
Claims (5)
1. the anti-brownization disinfectant of plant tissue culture explant, is characterized in that: adopt reproducibility bactericide soaking disinfection explant.
2. explant disinfectant as claimed in claim 1, is characterized in that: described reproducibility disinfectant is tetra methylol sulfuric acid phosphonium and 2-benzyl-4-chlorophenol.
3. disinfectant as claimed in claim 2, it is characterized in that: described tetrakis hydroxymetyl phosphonium sulfuric is directly dissolved in water, 2-benzyl-4-chlorophenol is dissolved in absolute ethyl alcohol and Tween-20,2-benzyl-4-chlorophenol and ethanol, Tween-20 weight (g) volume (ml), than being 1: 3: 5, are then dissolved in water.
4. disinfectant as claimed in claim 2, is characterized in that: tetrakis hydroxymetyl phosphonium sulfuric concentration 0.5-1.0%, soak time 60-90 minute, repeatedly stirs or concussion during this time.2-benzyl-4-chlorophenol concentration 0.5%, soak time 30-60 minute, repeatedly stirs or concussion during this time.
5. disinfectant as claimed in claim 4, explant soaks through tetrakis hydroxymetyl phosphonium sulfuric, sterile water washing 2 times, then soak through 2-benzyl-4-chlorophenol, taking-up is blotted residual solution with aseptic paper, directly inoculation.
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| CN108669097A (en) * | 2018-06-28 | 2018-10-19 | 合肥艾飞新材料有限公司 | A kind of preparation method of disinfectant use in agriculture |
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